EP3543251B1 - Fester träger mit igg-bindendem peptid und verfahren zur trennung von igg - Google Patents

Fester träger mit igg-bindendem peptid und verfahren zur trennung von igg Download PDF

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EP3543251B1
EP3543251B1 EP17870668.5A EP17870668A EP3543251B1 EP 3543251 B1 EP3543251 B1 EP 3543251B1 EP 17870668 A EP17870668 A EP 17870668A EP 3543251 B1 EP3543251 B1 EP 3543251B1
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Prior art keywords
igg
peptide
solid
phase support
column
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French (fr)
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EP3543251A1 (de
EP3543251A4 (de
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Yuji Ito
Seiichi Uchimura
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Daicel Corp
Kagoshima University NUC
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Daicel Corp
Kagoshima University NUC
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
    • B01D15/38—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving specific interaction not covered by one or more of groups B01D15/265 - B01D15/36
    • B01D15/3804—Affinity chromatography
    • B01D15/3809—Affinity chromatography of the antigen-antibody type, e.g. protein A, G, L chromatography
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/06—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies from serum
    • C07K16/065—Purification, fragmentation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K17/00—Carrier-bound or immobilised peptides; Preparation thereof
    • C07K17/02—Peptides being immobilised on, or in, an organic carrier
    • C07K17/08—Peptides being immobilised on, or in, an organic carrier the carrier being a synthetic polymer
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K17/00—Carrier-bound or immobilised peptides; Preparation thereof
    • C07K17/02—Peptides being immobilised on, or in, an organic carrier
    • C07K17/10—Peptides being immobilised on, or in, an organic carrier the carrier being a carbohydrate
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K17/00—Carrier-bound or immobilised peptides; Preparation thereof
    • C07K17/02—Peptides being immobilised on, or in, an organic carrier
    • C07K17/06—Peptides being immobilised on, or in, an organic carrier attached to the carrier via a bridging agent
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology

Definitions

  • the present invention relates to a solid-phase support including an IgG-binding peptide; an IgG separation column including the solid-phase support; a kit including the solid-phase support or the column; and a method for purifying IgG using the solid-phase support or the column.
  • IgG antibodies are now one of biopharmaceuticals attracting the most attention.
  • antibody drugs particularly IgG antibodies, have been used in the pharmaceutical field, increasingly gaining importance in industrial and pharmaceutical applications thereof.
  • protein A columns play a central role, and many manufacturers of antibody drugs have adopted purification systems centered on protein A columns.
  • Protein A is a protein derived from bacteria and is highly immunogenic after administration to the human body, and endotoxin contamination is a concern. Accordingly, to prevent contamination with unfavorable substances, protein A is required to be highly purified as an affinity ligand used for the purification of pharmaceuticals, such as IgG. This causes an increase in the cost of protein A columns used for the purification of pharmaceuticals. Therefore, development of a new affinity column replacing Protein A is expected.
  • Patent Document 1 WO 2013/027796
  • the present invention provides an IgG-binding peptide which can be used for the purification of IgG and has excellent stability, for example, alkali stability.
  • the present invention also provides a method for purifying IgG using the IgG-binding peptide.
  • the present inventor found that stability of a peptide is remarkably improved by cross-linking sulfide groups in cysteine residues in a peptide by a linker having a specific structure, thereby accomplishing the present invention.
  • the invention also provides an IgG separation column, comprising the solid-phase support of the present invention.
  • kits for purifying IgG including the solid-phase support of the invention or the IgG separation column of the invention.
  • Another aspect of the invention is a method for purifying IgG, including:
  • the peptide included in the solid-phase support of the present invention has improved stability by cross-linking sulfide groups in cysteine residues by a linker having a specific structure. Accordingly, the IgG binding capacity of the solid-phase support of the present invention is not likely to be diminished due to a process, such as an alkaline washing step, and thus the solid-phase support of the present invention can be used for an efficient purification of IgG.
  • Solid-phase support including IgG-binding peptide
  • an embodiment of the present invention relates to a solid-phase support including an IgG-binding peptide.
  • solid-phase support inorganic supports, such as glass beads and silica gel; organic supports composed of a synthetic polymer, such as cross-linked polyvinyl alcohol, cross-linked polyacrylate, cross-linked polyacrylamide, and cross-linked polystyrene; and polysaccharide, such as crystalline cellulose, cross-linked cellulose, cross-linked agarose, and cross-linked dextran; as well as composite supports obtained from combinations thereof, such as organic-organic and organicinorganic supports.
  • hydrophilic supports are preferred due to their relatively low non-specific adsorption and good selectivity for the IgG-binding peptide.
  • Hydrophilic supports herein refer to supports with a water contact angle of 60° or less when a compound forming the support is produced into a flat plate shape.
  • Representative examples of such supports include those composed of polysaccharide, such as cellulose, chitosan, and dextran; polyvinyl alcohol; a saponified ethylene-vinyl acetate copolymer; polyacrylamide; polyacrylic acid; polymethacrylic acid; polymethyl methacrylate; polyacrylic acid-grafted polyethylene; polyacrylamide-grafted polyethylene; and glass.
  • the solid-phase support may be in any form, such as beads, fibers, particles, membranes (including hollow fibers), and gels; and a solid-phase support in any form can be selected.
  • Solid-phase supports in a bead form are particularly preferably used for ease of preparing a support having a specific exclusion limit molecular weight.
  • Solid-phase supports in a bead form having an average particle size ranging from 10 to 2500 ⁇ m are easy to use; and in particular, those having an average particle size ranging from 25 ⁇ m to 800 ⁇ m are preferred for ease of immobilization reaction of the IgG-binding peptide.
  • examples of the solid-phase support include magnetic beads, glass beads, polystyrene beads, silica gel beads, and polysaccharide beads.
  • a functional group which can be used for the immobilization reaction of the IgG-binding peptide, on the surface of the solid-phase support is advantageous for immobilizing the IgG-binding peptide.
  • these functional groups include a hydroxy group, an amino group, an aldehyde group, a carboxyl group, a thiol group, a silanol group, an epoxy group, a succinimide group, an N-hydroxysuccinimide group, an acid anhydride group, and an iodoacetyl group.
  • a commercially available solid-phase support can be also used.
  • a commercially available products can be exemplified by GCL2000 and GC700, which are porous cellulose gels; Sephacryl S-1000, obtained by covalently cross-linking an allyl dextran and methylenebisacrylamide; Toyopearl, an acrylate-based support; Sepharose CL4B, an agarose-based cross-linked support; Eupergit C250L, a polymethacrylamide activated with epoxy groups; and an NHS-activated prepacked column containing a sepharose support activated with NHS groups.
  • the present embodiment is not limited to only these supports or activated supports.
  • the solid-phase supports described above may be each used alone or in a mixture of any two or more types.
  • the solid-phase support desirably has a large surface area and thus a large number of pores with a suitable size; that is, the solid-phase support is preferably porous.
  • the IgG-binding peptide described in the present specification is immobilized on the solid-phase support.
  • the peptide can be immobilized by a method known to those skilled in the art, for example, by physical adsorption method, covalent bonding method, and ionic bonding method. It is preferable to achieve the immobilization, for example, by covalently binding the N-terminal amino group of the IgG-binding peptide to the solid-phase support directly or via a spacer. It is more preferable to immobilize the peptide via a hydrophilic spacer in order to enhance separation efficiency by reducing steric hindrance of the IgG-binding peptide, and in order to suppress non-specific binding.
  • the hydrophilic spacer is not particularly limited, but it is preferable to use, for example, a derivative of a polyalkylene oxide, which has both terminals substituted with functional groups, such as a carboxyl group, an amino group, an aldehyde group, and an epoxy group.
  • a derivative of a polyalkylene oxide which has both terminals substituted with functional groups, such as a carboxyl group, an amino group, an aldehyde group, and an epoxy group.
  • a method and conditions for immobilizing the IgG-binding peptide to be introduced to the solid-phase support and an organic compound to be used as the spacer are not particularly limited, but they are exemplified by methods commonly employed to immobilize a protein and a peptide on a support.
  • One example is a method including: subjecting a support to a reaction with a compound containing an amino group, a compound containing an N-hydroxysuccinimidyl group, cyanogen bromide, epichlorohydrin, diglycidyl ether, tosyl chloride, tresyl chloride, hydrazine, or the like, to activate the support (by transforming a functional group into a group that is more reactive with an IgG-binding peptide than the functional group the support originally has); and then subjecting the support to a reaction with an IgG-binding peptide to immobilize the peptide thereto.
  • Another immobilization method includes adding a condensation reagent, such as carbodiimide, or a reagent having a plurality of functional groups in a molecule, such as glutaraldehyde, into a system in which a support and an IgG-binding peptide are present, to condense or cross-link them, thereby immobilizing them. It is more preferable, however, to utilize an immobilization method where the IgG-binding peptide is not easily released from the solid-phase support during sterilization or use of the solid-phase support.
  • a condensation reagent such as carbodiimide
  • a reagent having a plurality of functional groups in a molecule such as glutaraldehyde
  • the solid-phase support including the IgG-binding peptide described in the present specification can be loaded into a chromatography column, and the like, and used to purify or separate human IgG.
  • IgG-binding peptide included in the solid-phase support of an embodiment of the present invention will be described in detail below.
  • IgG used in the present specification refers to IgG of mammals, for example, primates, such as humans and chimpanzees; experimental animals, such as rats, mice, and rabbits; livestock animals, such as pigs, cows, horses, sheep, and goats; and pet animals, such as dogs and cats; preferably IgG of human (IgG1, IgG2, IgG3, or IgG4).
  • IgG in the present specification is more preferably human IgG1, IgG2, or IgG4, or rabbit IgG, and particularly preferably human IgG1, IgG2, or IgG4.
  • the peptide used in the present invention comprises the amino acid sequence GPDCAYHRGELVWCTFH (SEQ ID NO:31).
  • the IgG-binding peptide described in the present specification has at least two cysteine (C) residues positioned separately from each other in each amino acid sequence, and sulfide groups of the cysteine residues are connected via a linker selected from the group consisting of linkers represented by the following formula:
  • a method for preparing the peptide having the linker is not particularly limited.
  • a peptide having a linker represented by the following formula: can be obtained, for example, by a method including mixing a peptide containing two cysteine residues, and a compound having, at the wavy line portions of the linker, reactive functional groups (for example, halogen groups and imidazole groups) which are to be involved in the cross-linking reaction, for example, under acidic conditions.
  • the halogen groups in the compound above are selected preferably from the group consisting of F, Cl, Br, and I; and more preferably from the group consisting of Cl, Br, and I.
  • the halogen groups are preferably the same to each other, and more preferably all the halogen groups are Cl.
  • the conditions for the mixing step in the preparation method are not particularly limited as long as they can proceed the linking reaction between and the cysteine residues of the peptide to occur.
  • the reaction can be carried out by mixing the peptide and the compound above in a suitable buffer at room temperature (for example, about 15°C to 30°C) or at low temperature.
  • the mixing step may be carried out by adding an appropriate amount of a base (or an alkali) that promotes the linking reaction, for example, a weak basic inorganic or organic compound (for example, guanidium chloride, sodium bicarbonate, and diethylamine).
  • a mixing ratio of the peptide and the compound in the mixing step of the preparation method is not particularly limited.
  • a molar ratio of the peptide and the compound can be, for example, from 1:0.2 to 1:10, preferably from 1:0.5 to 1:5 or from 1: 1 to 1:2.
  • a mixing time (reaction time) of the mixing step is not limited as long as the linking reaction can occur between the cysteine residues in the peptide, but it can be, for example, from 1 min to 5 hours, and preferably from 10 min to 2 hours or from 15 min to 1 hour.
  • the method may further include separating impurities, such as, for example, an unreacted peptide or an unreacted compound, from the mixture after the mixing step, to purify a peptide of which cysteine residues are linked together.
  • impurities such as, for example, an unreacted peptide or an unreacted compound
  • the step can be carried out by a known method in the art, for example, chromatography, such as gel filtration chromatography, ion-exchange column chromatography, affinity chromatography, reverse-phase column chromatography, and HPLC.
  • the IgG-binding peptide described in the present specification may be modified, for example, by N-terminal PEGylation (polyethylene glycol addition) and C-terminal amidation, for example, to improve stability thereof.
  • the number of PEG molecules for the PEGylation is not particularly limited, and, for example, from 1 to 50 molecules, from 1 to 20 molecules, from 2 to 10 molecules, from 2 to 6 molecules, or 4 molecules of PEG can be added.
  • the IgG-binding peptide described in the present specification may be multimerized.
  • "multimerization" of the IgG-binding peptide means that two or more molecules of the IgG-binding peptide are linked via a covalent bond.
  • the multimer of the IgG-binding peptide may be, for example, from a dimer to a hexamer, from a dimer to a pentamer, from a dimer to a tetramer, from a dimer to a trimer, and preferably a dimer.
  • the multimer of the peptide may include a spacer between the peptides.
  • the multimerization can be achieved by a method known to those skilled in the art, for example, by linking N-terminal amino groups of two or more molecules of the IgG-binding peptide via a spacer.
  • the type of the spacer is not particularly limited, but examples thereof include an amino acid such as aspartic acid and glutamic acid, which have carboxyl groups at both termini; and a derivative of a polyalkylene oxide, which is substituted at both termini with functional groups, such as a carboxyl group, an aldehyde group, an epoxy group, and an N-hydroxysuccinimidyl group.
  • the IgG-binding peptide described in the present specification has binding affinity for human IgG which may be at least about 10 times, preferably at least about 50 times, and more preferably at least about 200 times as high as that for other human immunoglobulins (IgA, IgE, and IgM).
  • the dissociation constant (Kd) for binding between the peptide described in the present specification and human IgG can be determined by surface plasmon resonance spectral analysis (for example, using a BIACORE system), and Kd is, for example, less than 1 ⁇ 10 -1 M, less than 1 ⁇ 10 -3 M, preferably less than 1 ⁇ 10 -4 M, and more preferably less than 1 ⁇ 10 -5 M.
  • the IgG-binding peptide described in the present specification can bind to the Fc domain of IgG.
  • the peptide described in the present specification can be produced by a peptide synthesis method, such as commonly used liquid phase peptide synthesis and solid phase peptide synthesis, and also by peptide synthesis with an automated peptide synthesizer ( Kelley et al., Genetics Engineering Principles and Methods, Setlow, J.K. eds., Plenum Press NY. (1990) Vol.12, p.1-19 ; Stewart et al., Solid-Phase Peptide Synthesis (1989) W.H. Freeman Co. ; Houghten, Proc. Natl. Acad. Sci.
  • the peptide may be produced by a method, such as a genetic recombination method and a phage display method, using a nucleic acid encoding the peptide described in the present specification.
  • the target peptide can be produced by incorporating DNA encoding the amino acid sequence of the peptide described in the present specification into an expression vector, introducing the resulting vector into a host cell, and then culturing the host cell.
  • the peptide thus produced can be collected or purified by an ordinary method, for example, chromatography, such as gel filtration chromatography, ion-exchange column chromatography, affinity chromatography, reverse-phase column chromatography, and HPLC; ammonium sulphate fractionation, ultrafiltration, and an immunoadsorption method.
  • chromatography such as gel filtration chromatography, ion-exchange column chromatography, affinity chromatography, reverse-phase column chromatography, and HPLC
  • ammonium sulphate fractionation ultrafiltration, and an immunoadsorption method.
  • amino acids are prepared by protecting functional groups of each amino acid (whether natural or unnatural) other than the ⁇ -amino group and the ⁇ -carboxyl group to be bound, and then the ⁇ -amino group and the ⁇ -carboxyl group of each amino acid are subjected to a reaction to form a peptide bond therebetween.
  • the carboxyl group of an amino acid residue positioned at the C-terminus of the peptide is bound to a solid phase in advance via a suitable spacer or linker.
  • the protecting group at the amino terminus of the dipeptide thus obtained is selectively removed, and a peptide bond with the ⁇ -carboxyl group of the next amino acid is formed.
  • Such an operation is continuously carried out to produce a peptide having protected side groups, and finally, all the protecting groups are removed, and the peptide is detached from the solid phase.
  • Types of the protecting group, a protection method, and a peptide binding method are detailed in the above documents.
  • Production by a genetic recombination method may include, for example, inserting DNA that encodes the peptide described in the present specification into a suitable expression vector, introducing the resulting vector into a suitable host cell, culturing the cell, and collecting the target peptide from the inside of the cell or from the extracellular fluid.
  • a suitable expression vector examples include, but are not limited to, a vector, such as a plasmid, a phage, a cosmid, a phagemid, and a virus.
  • the plasmid vector include, but are not limited to, an E.
  • coli-derived plasmid such as pET22b(+), pBR322, pBR325, pUC118, pUC119, pUC18, pUC19, and pBluescript
  • Bacillus subtilis-derived plasmid such as pUB110 and pTP5
  • yeast-derived plasmid such as YEp13 and YCp50
  • phage vector examples include, but are not limited to, a T7 phage display vector (such as T7Select10-3b, T7Select1-1b, T7Select1-2a, T7Select1-2b, and T7Select1-2c (Novagen)) and a ⁇ phage vector (such as Charon4A, Charon21A, EMBL3, EMBL4, ⁇ gt10, ⁇ gt11, ⁇ ZAP, and ⁇ ZAPII).
  • T7 phage display vector such as T7Select10-3b, T7Select1-1b, T7Select1-2a, T7Select1-2b, and T7Select1-2c (Novagen)
  • a ⁇ phage vector such as Charon4A, Charon21A, EMBL3, EMBL4, ⁇ gt10, ⁇ gt11, ⁇ ZAP, and ⁇ ZAPII).
  • virus vector examples include, but are not limited to, an animal virus, such as a retrovirus, an adenovirus, an adenoassociated virus, a vaccinia virus, and Sendai virus; and an insect virus, such as a baculovirus.
  • cosmid vector examples include, but are not limited to, Lorist 6, Charomid 9-20, and Charomid 9-42.
  • a known phagemid vector examples include, but are not limited to, pSKAN, pBluescript, pBK, and pComb3H.
  • a vector can include, for example, a regulatory sequence so as to enable the expression of the target DNA, a selection marker to select a vector containing the target DNA, and a multicloning site to insert the target DNA.
  • a regulatory sequence includes, for example, a promoter, an enhancer, a terminator, an S-D sequence or a ribosome binding site, a replication origin, and a poly A site.
  • the selection marker for example, an ampicillin resistant gene, a neomycin resistant gene, a kanamycin resistant gene, and a dihydrofolate reductase gene can be used.
  • the host cell into which the vector is to be introduced is, for example, a bacterium, such as E.
  • coli and Bacillus subtilis a yeast cell; an insect cell; an animal cell (such as a mammalian cell), and a plant cell.
  • transformation or transfection into these cells include a calcium phosphate method, an electroporation method, a lipofection method, a particle bombardment method, and a PEG method.
  • the transformed cells are cultured in accordance with a common method used for culturing host organisms.
  • a culture medium for microorganisms such as E. coli and yeast cells, contains a substance, such as a carbon source, a nitrogen source, and inorganic salts, that can be utilized by the host microorganisms.
  • the host organisms to secrete the peptide produced by expression to the outside of the cell.
  • This can be achieved by binding a DNA that encodes a peptide sequence enabling the secretion of the peptide from the cell to the 5'-terminal side of the DNA that encodes the target peptide.
  • a fusion peptide that has migrated to the cell membrane is cleaved by signal peptidase, and thus the target peptide is secreted and released into the medium.
  • an embodiment of the present invention relates to a column for separating an IgG, preferably a human IgG, that includes the solid-phase support including the IgG-binding peptide.
  • the IgG separation column encompasses a column, such as a chromatography column and a high-performance liquid chromatography (HPLC) column, for purification or separation of IgG.
  • the size of the column is not particularly limited, and it can be varied depending on, for example, the intended use, such as for analysis, purification, or fractionation; the amount of a sample to be applied (loaded) or injected, and the length or the inner diameter of the column.
  • the column may be made of a material commonly used for a column, such as metal, plastic, and glass.
  • the column can be produced by densely filling a column with the solid-phase support of an embodiment of the present invention (which may be in either dry or wet state).
  • an embodiment of the present invention relates to a kit for purifying IgG, preferably human IgG, that includes the solid-phase support including the IgG-binding peptide.
  • the kit of an embodiment of the present invention may include at least one of: a manual for use describing analytical procedures and purification procedures for human IgG, a reagent and a buffer necessary for purification, or a column to be filled with the solid-phase support.
  • an embodiment of the present invention relates to a method for purifying IgG, preferably human IgG, including: binding IgG to the solid-phase support or the IgG separation column; and eluting the bound IgG to collect the IgG.
  • the binding step can be carried out by a method known to those skilled in the art.
  • the solid-phase support or the IgG separation column are equilibrated with a suitable buffer, and then a liquid containing IgG is applied thereto at low temperature from 0°C to room temperature, preferably from 0°C to about 10°C, more preferably at about 4°C, to bind the IgG to the peptide on the solid-phase support.
  • the binding step can be carried out by applying a liquid containing serum and IgG to the column, using a buffer having a pH in the neutral range, for example, pH from 6.0 to 7.5.
  • the elution step can be also carried out by a method known to those skilled in the art.
  • the IgG may be eluted by feeding a buffer having a pH in the acidic range, for example, pH from 2 to 4 (for example, 0.2 M glycine-HCl buffer or 20 mM citrate buffer, containing 0.3 M NaCl, from pH 3.5 to pH 2.5), through the column, or by competitive elution using the IgG-binding peptide.
  • a buffer having a pH in the acidic range for example, pH from 2 to 4 (for example, 0.2 M glycine-HCl buffer or 20 mM citrate buffer, containing 0.3 M NaCl, from pH 3.5 to pH 2.5), through the column, or by competitive elution using the IgG-binding peptide.
  • the solid-phase support or the column can be regenerated and reused in the binding step by washing the support or the column with an alkaline solution, such as a sodium hydroxide solution, a potassium hydroxide solution, and a potassium hydroxide solution (for example, 0.1 M sodium hydroxide solution).
  • an alkaline solution such as a sodium hydroxide solution, a potassium hydroxide solution, and a potassium hydroxide solution (for example, 0.1 M sodium hydroxide solution).
  • an alkaline solution such as a sodium hydroxide solution, a potassium hydroxide solution, and a potassium hydroxide solution (for example, 0.1 M sodium hydroxide solution).
  • an alkaline solution such as a sodium hydroxide solution, a potassium hydroxide solution, and a potassium hydroxide solution (for example, 0.1 M sodium hydroxide solution).
  • the degree of alkalinity of the solution will be easily determined by those skilled in the art. Accordingly, the method of an embodiment of the present invention can optionally include regenerating the
  • IgG is collected can be determined, for example, by identification of molecular weight by electrophoresis, and optionally subsequent Western blotting using an anti-IgG antibody.
  • the electrophoresis may be carried out by SDS-PAGE using a 5 to 20% acrylamide gradient gel, and Western blotting can be carried out by transferring proteins after electrophoresis to a PVDF membrane, followed by blocking with skimmed milk, and then detecting the IgG with a goat anti-IgG ⁇ chain antibody and an HRP-labeled mouse anti-goat IgG antibody.
  • the method of an embodiment of the present invention is useful for obtaining an IgG-rich fraction in a step of purifying IgG from an IgG-containing product produced by various methods.
  • column chromatography such as affinity chromatography and HPLC.
  • protein purification techniques for purification of IgG, in addition to such chromatography, commonly used protein purification techniques, for example, chromatography, such as gel filtration chromatography, ion-exchange column chromatography, and reverse-phase column chromatography; ammonium sulphate fractionation; and ultrafiltration can be combined as appropriate.
  • an intramolecular S-S bond was formed in an aqueous solution having a pH of 8.5 under oxidative conditions, and a peptide having the intramolecular S-S bond was purified by reverse-phase HPLC using a gradient elution with 10% to 60% acetonitrile containing 0.1% TFA at a flow rate of 1.0 mL/min.
  • Affinity analysis of the purified IgG-binding peptide was carried out according to the following method. Equal amounts of 0.4 M 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) and 0.1 M sulfo-N-hydroxysuccinimide (sulfo-NHS) were mixed and injected onto a CM5 sensor chip, which was set in BIAcoreT200 (GE healthcare), at a flow rate of 10 ⁇ L/mL for 7 min to activate the sensor chip, and then the IgG was immobilized on the sensor chip under conditions of pH 4.0 (10 mM Na acetate) so as to give an immobilized amount of 4000 to 5000 in RU value.
  • EDC 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide
  • sulfo-NHS sulfo-N-hydroxysuccinimide
  • the binding reaction was monitored by injecting the peptide in a concentration from 10 nM to 2 ⁇ M at a flow rate of 50 ⁇ L/mL for 180 sec while using an HBS-EP buffer (0.01 M HEPES, 0.15 M NaCl, 0.005% Tween 20, 3 mM EDTA, pH 7.0), and then the dissociation reaction was measured by washing with the buffer for 600 sec. Binding parameters were analyzed using BIAevalution T100 software.
  • Results of the affinity measurement are shown in Table 1 below. Results in Table 1 show that all the peptides can bind to IgG and thus can be used for purification of an antibody.
  • Table 1 Sequence of peptide SEQ ID NO: ka kd KD (nM) 1:1 binding Equilibriu m value DCAYHXaa1GELVWCT-NH 2 1, wherein Xaal is R 4.57E+05 0.0248 54 64.5 GPRCAYHXaa1GELVWCSFH-NH 2 4, wherein Xaal is R 8.40E+05 0.0353 42 56 GPDCAYHXaa1GELVWCTFH-NH 2 2, wherein Xaal is R 1.57E+06 0.0144 9.1 10 GPDCAYHXaa1GELVWCTFH-NH 2 2, wherein Xaal is L 1.7E+05 0.014 8.1 - GPDCAYHXaa1GELVWCTFH-NH 2 2, wherein Xaal is K 1.25E+
  • 1,3-dichloro-2-propanone 2.9 mg, 23.4 ⁇ mol, 1.5 molar equivalent
  • N-terminally PEG4-modified and C-terminally amidated peptide (Peptide a, according to the invention) including an amino acid sequence represented by GPDCAYH(Xaal)GELVWCTFH (SEQ ID NO:2, wherein Xaal was arginine and the C-terminus was amidated), in which sulfide groups in the two outermost cysteine residues were connected via a linker represented by the following formula:
  • This peptide was measured for affinity for IgG as in Example 1, and Kd was revealed to be 1 ⁇ M.
  • Example 3 Separation of human serum-derived ⁇ -globulin
  • a dichloroaceton-cross-linked peptide was immobilized to an NHS-activated prepacked column (GE Healthcare) to carry out various evaluations to assess whether the dichloroaceton-cross-linked peptide can be used as an affinity ligand for human antibody purification.
  • the peptide-immobilized column was prepared by the following procedures. A syringe was used to feed a solution.
  • the prepared peptide-immobilized column was connected to a liquid chromatography system AKTAexplore (GE Healthcare) and equilibrated with the adsorption solution. Then, a 1 mg/mL solution of human serum-derived ⁇ -globulin (Wako) dissolved in the adsorption solution was fed to the column at a flow rate of 1 mL/min for 1 min. Furthermore, the column was washed with the adsorption solution, and the adsorbed components were eluted by feeding an acidic elution solution (20 mM citric acid, pH 2.5). The elution of proteins from the column was detected by absorbance at 280 nm. Experimental results are illustrated in FIG. 1 .
  • Example 2 As illustrated in FIG. 1 , along with the decrease of pH, elution of the human serum-derived ⁇ -globulin adsorbed in the column was confirmed, revealing that the peptide prepared in Example 2 can be used as a ligand for an affinity column.
  • Example 3 To the column prepared by the same method as in Example 3, 1 mg of human-derived serum ⁇ -globulin was fed to adsorb the globulin thereto. The column was washed with the adsorption solution, and then 2.5 mL of a 0.4 mg/mL solution of Peptide a prepared in Example 2 dissolved in the adsorption solution was fed to the column. Each fraction fractionated in 0.5 mL was subjected to SDS-PAGE under reductive conditions in accordance with an ordinary method. Proteins were detected by CBB staining. For comparison, similar operations were carried out also for citric acid elution.
  • Example 3 To a 1-mg peptide-immobilized 1-mL column prepared by the same method as in Example 3, 5 mL of 0.1 M sodium hydroxide solution was fed. The column was then washed with the adsorption solution and DBC was measured at a flow rate of 1 mL/min as in Example 5. Subsequently, a cycle of the treatment with the sodium hydroxide solution and the DBC measurement was repeated five times to evaluate the alkali resistance. The variation rate of DBC was determined based on the DBC before the sodium hydroxide treatment as 100%.
  • Example 2 As Comparative Example 1, a column with 1 mg of a peptide cross-linked by a disulfide bond immobilized thereon was prepared, and the alkali resistance evaluation was carried out as in Example 5.
  • Example 5 Results from Example 5 and Comparative Example 1 are illustrated in FIG. 4 , and measured values are summarized in Table 3.
  • Table 3 DBC 10% (% NaOH washing cycle 0) DBC 10% (mg/mL) (% NaOH washing cycle) Comparative Example 1
  • Example 5 Comparative Example 1
  • Example 5 0 100.0 100.0 14.0 4.8 1 98.6 100.0 13.8 4.8 2 95.0 102.1 13.3 4.9 3 92.9 100.0 13.0 4.8 4 88.6 100.0 12.4 4.8 5 86.4 97.9 12.1 4.7
  • the peptide cross-linked by a disulfide bond showed a decrease in DBC to 86.4% by the five sodium hydroxide treatments (Comparative Example 1).
  • the dichloroaceton cross-linked peptide showed no decrease in DBC, revealing that the peptide has high alkali resistance.
  • the peptide included in the solid-phase support of the present invention has improved stability by cross-linking sulfide groups in cysteine residues by a linker having a specific structure. Accordingly, the IgG binding capacity of the solid-phase support of the present invention is not likely to be diminished due to a process, such as an alkaline washing step, and thus the solid-phase support of the present invention can be used for an efficient purification of IgG and production of IgG that is used also as a pharmaceutical.

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Claims (8)

  1. Festphasenträger mit einem darauf immobilisierten Peptid, wobei das Peptid in der Lage ist, an menschliches IgG zu binden,
    wobei das Peptid die folgende Aminosäuresequenz umfasst:
    GPDCAYHRGELVWCTFH (SEQ ID NO: 31) und
    Sulfidgruppen in den beiden äußersten Cysteinresten in dem Peptid über einen Linker der Formel:
    Figure imgb0007
     verbunden sind.
  2. Festphasenträger gemäß Anspruch 1, wobei der N-Terminus des Peptids PEGyliert ist.
  3. Festphasenträger gemäß Anspruch 1 oder 2, wobei der C-Terminus des Peptids amidiert ist.
  4. Festphasenträger gemäß einem der Ansprüche 1 bis 3, wobei das Peptid multimerisiert ist, wobei das Multimer des Peptids vorzugsweise einen Spacer zwischen den Peptiden umfasst.
  5. Festphasenträger gemäß einem der Ansprüche 1 bis 4, umfassend einen Spacer zwischen dem Peptid und der Festphase.
  6. IgG-Trennsäule, umfassend den in einem der Ansprüche 1 bis 5 beschriebenen Festphasenträger.
  7. Kit zum Reinigen von IgG, umfassend den in einem der Ansprüche 1 bis 5 beschriebenen Festphasenträger oder die in Anspruch 6 beschriebene IgG-Trennsäule.
  8. Verfahren zum Reinigen von IgG, umfassend:
    Binden von IgG an den in einem der Ansprüche 1 bis 5 beschriebenen Festphasenträger oder die in Anspruch 6 beschriebene IgG-Trennsäule und
    Eluieren des gebundenen IgG, um das IgG zu gewinnen.
EP17870668.5A 2016-11-18 2017-11-17 Fester träger mit igg-bindendem peptid und verfahren zur trennung von igg Active EP3543251B1 (de)

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